Introduction
Bone tissue is a mineralized connective tissue in perpetual renewal thanks to the bone remodeling process. Therefore, it manages to spontaneously repair traumatic or non-traumatic lesions. Unfortunately, in the case of large defects, bone loses the ability to renew itself, implying that surgeons must use other strategies to regenerate or replace damaged bone. Autologous bone graft is the most popular strategy of bone repair. However, this strategy has some limits such as severe pain, limited reserves in body and risk of necrosis and nerve damage at the donor site. Therefore, research has been directed toward the development of orthopaedic biomaterials such as implants and bone filling materials. These biomaterials are the most implanted devices representing 52% of implantations [1]. Despite the great success of these biomaterials, failure of bone-implant interface (lack of osteointegration ) leading to implants loosening remains the main drawback. Accordingly, this project objective is to enhance the osteointergation of orthopaedic implants in order to increase their long-term performance which is restricted to 10-15 years with the currently available materials [1].
Various strategies have been investigated to enhance the osteointegration process by inducing bone formation around orthopaedic implants. Two main schemes are well documented: the first is the chemical surface modification of biomaterial, which consists in grafting proteins or mimetic peptides. The second scheme is the physical surface modification of biomaterial, which involves the surface topography modification such as roughness, porosity, micropatterning either at the micrometer or nanometer scale. The original approach of this project is to combine these two modification strategies in order to mimick both the topographical and the chemical features of the extra cellular matrix (ECM) of the bone tissue. Briefly, two bioactive peptides were grafted on the surface of a model biomaterial. The first was the RGD peptide, which is well known to interact with cells through their integrin receptors, therefore facilitating cell spreading and adhesion on biomaterial surfaces. The second was the BMP-2 mimetic peptide, recently discovered by our laboratory to induce bone formation through the differentiation of Bone Mesenchymal Stem Cells (BMSCs) into mature osteoblasts capable of synthetizing a mineralized ECM [2]. In addition, a better expression of Runx2 (osteoblastic marker) was demonstrated in the case of materials functionalised with RGD and BMP2 mimetic peptides compared to materials just functionalised with BMP2 mimetic peptide [2]. Furthermore, RGD and BMP-2 mimetic peptides were not grafted randomly but were instead distributed on the surface as micro-patterns because such surface topography is known to direct stem cells toward osteoblastic lineage [3].

Materials and Methods

The borosilicate glass was used as biomaterial since it is widely commercialized in biomedical industry as an artificial hip joints, bone cements and dental composite materials. Aminated borosilicate glasses (NH2-borosilicate glass) were purchased from Schott in order to further graft the peptides of interest. SMPB crosslinker was used due to its ability to react at one hand by its succinimidyl function with borosilicate glass amines and at the other hand by its maleimide function with the terminal cysteine of RGD and BMP-2 mimetic peptides (Figure1). These two peptides were grafted in the form of desired micropatterns by using the maskless photolithography (Figure2). The designed peptides micropatterns corresponded to square, rectangle or triangle with a constant surface area of 50 µm². Surface characterization was achieved using X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) in order to assess the surface chemistry and topography of the material. Micropattern visualization was achieved using fluorescent peptides RGD-TAMRA and BMP-2-FITC which were thereafter observed through fluorescent microscopy.

Results

XPS analyses shown below evidenced the grafting efficiency since the nitrogen amount decreased after SMPB grafting from 3.8 % on initial aminated glass to 3.2 %. In the same way, the peptides grafting were confirmed by the lower detection of glass substrate (silicium) and the increase in nitrogen amount due to the peptides amino-groups.
Aminated glass: 42.1% C, 40.9% O, 3.8% N, 13.9% Si
SMPB grafted: 40.2% C, 42.3% O, 3.2% N, 14.3% Si
RGD grafted: 46.8% C, 34.5% O, 5.9% N, 12.7% Si
BMP-2 grafted: 39.1% C, 41.8% O, 5.4% N, 13.2% Si
As previousely mentioned, the peptides grafting as well as the micropatterning were visualized by fluorescence microscopy with the fluorescent peptides (figure 3). The spatial distribution of the two peptides designed as square, rectangular or triangular micropatterns were clearly evidenced and well defined as seen in Figure 3.

Discussion and Conclusion

The strategy of designing specific bioactive peptides micropatterned onto orthopaedic material surfaces seems to be promising for osteointegration enhancement. Our previous results in surface characterization evidenced that the peptides grafting as well as micropatterning were efficient. The next step of the project is to study the effect of peptides micropatterning on the behavior and the fate of BMSCs through different assessment assays such as cell proliferation, adhesion, differentiation as well as ECM mineralization.
Figure1: Representative scheme of peptides immobilization strategy on borosilicate glass surfaces.Figure2: Peptides micropatterning on borosilicate glass surfces using maskless photolithography.Figure3: Fluorescent images of surface micropatterning with fluorescent peptides: RGD-TAMRA (RED) and BMP 2–FITC (green).

Acknowledgements

Bilem Ibrahim was awarded of a PhD Doctoral Scholarship from NSERC CREATE Program in
Regenerative Medicine (http://www.ncprm.ulaval.ca).